Pyrophosphorolytic sequencing

ABSTRACT

A method for determining the sequence of a target nucleic acid, including steps of contacting a target nucleic acid with a polymerase to sequentially remove nucleotide triphosphates from the target nucleic acid, wherein the nucleotide triphosphates that are removed have a variety of different base moieties; and distinguishing the different base moieties for the nucleotide triphosphates that are removed. Also provided is a apparatus including a nanopore positioned in a fluid impermeable barrier to form a passage through which a nucleotide triphosphate can pass from a first fluid reservoir to a second fluid reservoir, and a reaction mix in the first fluid reservoir that includes a polymerase, target nucleic acid having two strands, and pyrophosphorolytic concentration of pyrophosphate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/286,447, filed May 23, 2014, which is based on, and claims thebenefit of, U.S. Provisional Application No. 61/827,175, filed May 24,2013, which is incorporated herein by reference.

BACKGROUND

This disclosure relates generally to nucleic acid analysis, and morespecifically to nucleic acid synthesis using nanopores.

Currently available commercial platforms for sequencing DNA arerelatively costly. These platforms use a ‘sequencing by synthesis’approach, so called because DNA polymers are synthesized while detectingthe addition of each monomer (i.e. nucleotide) to the growing polymerstructure. Because a template DNA strand strictly directs synthesis of anew DNA polymer, one can infer the sequence of the template DNA from theseries of nucleotide monomers that were added to the growing strandduring the synthesis. The ability to detect monomer additions isfacilitated by specially engineered variants of the biochemicalcomponents that normally carry out DNA synthesis in biological systems.These engineered components are expensive to make and are consumed inrelatively large amounts during sequencing by synthesis. Furthermore,monitoring the reaction uses relatively expensive hardware such aslasers, detection optics and complex fluid delivery systems. The mostsuccessful commercial platforms to date also require expensive reagentsand hardware to amplify the DNA templates before sequencing by synthesiscan even begin.

Other sequencing methods have been considered in order to reduce cost,increase throughput, and/or simplify the process. One of theseapproaches is based on threading a single strand of DNA through ananopore and identifying its sequence from the variation in the ioniccurrent flowing through the pore as the strand is threaded. Analternative to this ‘nanopore-strand’ sequencing approach is‘nanopore-exonuclease’ sequencing, which involves exonuclease catalyzedremoval of nucleotide monophosphates, one at a time, from a DNA strandand sequentially passing the released nucleotide monophosphates througha nanopore. However, the resulting variations in the ionic currentflowing through the nanopores are quite small and it is difficult todistinguish one nucleotide from another. Attempts have been made tomodify the DNA before digestion or to modify the nucleotidemonophosphates once they have been released. However despite theseefforts, nanopore-exonuclease sequencing has not yet been demonstratedat a commercially viable level to date.

Thus, there exists a need for more cost effective, rapid and convenientplatforms that provide an alternative to those currently available forsequencing nucleic acids. The present disclosure addresses this need andprovides other advantages as well.

BRIEF SUMMARY

The present disclosure provides a method for determining the sequence ofa target nucleic acid. The method can include the steps of (a) providinga target nucleic acid; (b) contacting the target nucleic acid with apolymerase to sequentially remove nucleotide triphosphates from thetarget nucleic acid, wherein the nucleotide triphosphates that areremoved have a variety of different base moieties; and (c)distinguishing the different base moieties for the nucleotidetriphosphates that are removed, thereby determining the sequence of thetarget nucleic acid.

In some embodiments a method for determining the sequence of a targetnucleic acid can be carried out using the steps of (a) providing atarget nucleic acid having two strands; (b) contacting the targetnucleic acid with a polymerase under conditions to sequentially removenucleotides from the first of the two strands by pyrophosphorolysis,thereby sequentially producing nucleotide triphosphates having a varietyof different base moieties; and (c) distinguishing the different basemoieties for the sequentially produced nucleotide triphosphates, therebydetermining the sequence of the target nucleic acid.

The present disclosure also provides a apparatus that includes (a) afluid impermeable barrier separating a first fluid reservoir from asecond fluid reservoir; (b) a nanopore positioned in the fluidimpermeable barrier to form a passage through which a nucleotidetriphosphate can pass from the first fluid reservoir to the second fluidreservoir; and (c) a reaction mix in the first fluid reservoir, thereaction mix comprising a polymerase, target nucleic acid having twostrands, and pyrophosphorylitic concentration of pyrophosphate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of a pyrophosphorolytic sequencing reaction usinga polymerase attached to a nanopore.

FIG. 2 shows a diagram of a pyrophosphorolytic sequencing reaction usinga polymerase attached to a nanopore and a template nucleic acid attachedto a fluid impermeable barrier.

FIG. 3. shows pyrophosphorolytic sequencing with membrane seeding ofmultiple nucleic acid templates.

DETAILED DESCRIPTION

The present disclosure provides a method of sequencing nucleic acids ina reverse fashion compared to standard sequencing by synthesis (SBS)techniques. In particular embodiments, the method of the presentdisclosure exploits a catalytic function of polymerase known aspyrophosphorolysis that is typically maligned as the culprit forunwanted artifacts in SBS techniques. Pyrophosphorolysis results in theremoval of nucleotide triphosphates from a nucleic acid strand by apolymerase, and as such is the reverse of the polymerization reactionthat drives standard SBS techniques.

Pyrophosphorolysis can be distinguished from exonuclease activity (whichis present in some polymerases), for example, based on the differentcatalytic mechanism for the two reactions, different active sites in thepolymerase structure where the two reactions occur, and the differentproducts for the reactions. Regarding the catalytic mechanism and activesite differences, it is known that exonuclease activity can be removedfrom many polymerase species by deletion of certain domains, whereaspyrophosphorolysis activity is believed to be catalyzed by the samedomain that catalyzes polymerization. Furthermore, the reaction cyclecatalyzed by exonuclease is the conversion of DNA_(n) (DNA of length n)to DNA_(n-1) (DNA that is one nucleotide shorter than DNA of length n)and a nucleotide monophosphate. In contrast, a cycle ofpyrophosphorolysis produces DNA_(n-1) and a nucleotide triphosphate fromDNA_(n) and pyrophosphate. Notably, pyrophosphate is consumed in apyrophosphorolysis reaction but is not consumed in an exonucleasereaction.

Particular embodiments of the pyrophosphorolytic sequencing methodsutilize nanopore detection. Nanopores can be used to sequentially detectthe nucleotide triphosphates that are released from a nucleic acid inorder to determine the sequence of the nucleic acid. Such embodimentsprovide a combination of advantages that are typically only partiallysatisfied by nanopore-exonuclease sequencing or nanopore-strandsequencing. Specifically, the pyrophosphorolytic sequencing methods ofthe present disclosure address some of the challenges incurred innanopore-exonuclease sequencing, namely low capture and detectionprobabilities, while retaining its main advantage over strandsequencing, namely single base resolution. This advantage derives fromthe fact that the affinity of nanopores for nucleotide monophosphates israther weak (on the order of micromolar affinity), even in the presenceof an am6-cyclodextrin adaptor that has been used to improve signal insome nanopore systems. See Clarke et al., Nat. Nanotechnol. April;4(4):265-70 (2009), which is incorporated herein by reference. Forsuccessful distinction of different nucleotide types in a sequencingcontext, nanomolar range affinity is desired. ATP has an affinity thatis in a good range, even without the use of an adaptor in the pore. SeeCheley et al., Chem. Biol. 9, 829-838 (2002), which is incorporatedherein by reference. Without wishing to be bound by theory, it ispostulated that the improved affinity of ATP over nucleotidemonophosphate is due to the triple negative charge carried by ATP, whichmay cause it to bind more strongly inside the nanopore. Furthermore, thetriple negative charge may aid capturing of the molecule in the presenceof an electric field, especially when the field is very weak, as is thecase outside of the nanopore where the nucleotides are actuallyreleased.

In addition to the enhanced capture and detection of nucleotidetriphosphates, there are a number of other advantages that can beprovided by embodiments set forth herein, such as the use of apolymerase as opposed to an exonuclease. Polymerases have been shown toform a good “fit” with nanopores for the purpose of nanopore-strandsequencing (Cherf et al., Nat. Biotech. 30:344-348 (2012); Manrao etal., Nat. Biotech. 30:349-353 (2012), each of which is incorporatedherein by reference). A similarly good fit is yet to be demonstratedwith exonucleases. Furthermore, the substrate for polymerases is doublestranded DNA which generally does not enter the nanopore. In contrast,single stranded DNA, the substrate for most exonucleases, can pose theproblem of entering and blocking the nanopore. Finally, unlike in eitherexonuclease-based sequencing or polymerase-based strand sequencing, therate of a pyrophosphorolytic sequencing reaction can be controlled bytuning the pyrophosphate concentration.

Terms used herein will be understood to take on their ordinary meaningunless specified otherwise. Examples of several terms used herein andtheir definitions are set forth below.

As used herein, the term “attached” is intended to mean connected byforces that prevent separation by diffusion. The term can includeconnections that are covalent or non-covalent in nature. For example twoproteins can be covalently attached through their primary sequence (e.g.a peptide linkage or protein fusion) or between their primary sequences(e.g. a disulfide linkage or chemical crosslink via amino acid Rgroups). Two proteins can be non-covalently attached, for example, viaone or more of hydrogen bonds, ionic bonds, van der Waals forces,hydrophobic bonds or the like.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context unambiguouslydictates otherwise.

As used herein, the term “exonuclease activity” is intended to meanhydrolysis of the phosphodiester bond that attaches a nucleotide to theend of a nucleic acid of length n to produce a nucleotide monophosphateand a nucleic acid of length n−1. The hydrolysis can occur at the bondthat attaches the 5′ nucleotide to the nucleic acid (i.e. 5′ to 3′exonuclease activity) or at the bond that attaches the 3′ nucleotide tothe nucleic acid (i.e. 3′ to 5′ exonuclease activity).

As used herein, the term “fluid impermeable barrier” is intended to meananything that prevents passage of a fluid. For example, a fluidimpermeable barrier can be present between two reservoirs such that afluid in the first reservoir is separated from the fluid in the secondreservoir, and the fluids do not mix. A fluid impermeable barrier caninclude a pore or opening that allows passage of a fluid that isotherwise obstructed by the remainder of the barrier. In particularembodiments, the fluid can be an aqueous fluid and the barrier can beimpermeable to aqueous fluids.

As used herein, the term “lacks exonuclease activity” is intended tomean having no measurable exonuclease activity. For example, apolymerase or other agent that lacks 3′ to 5′ exonuclease activity willhave no measurable 3′ to 5′ exonuclease activity. Similarly, apolymerase or other agent that lacks 5′ to 3′ exonuclease activity willhave no measurable 5′ to 3′ exonuclease activity. Polymerases known inthe art as “exo minus” (or “exo-”) whether naturally occurring orengineered are non-limiting examples of polymerases that lackexonuclease activity. Known variants include those that are 5′ to 3′ exominus and/or 3′ to 5′ exo minus.

As used herein, the term “nanopore” is intended to mean a small holethat allows passage of nucleotide triphosphates across an otherwiseimpermeable barrier. The barrier is typically an electrically insulatinglayer and the nanopore typically permits ions to flow from one side ofthe barrier to the other, driven by an applied potential. The nanoporepreferably permits nucleotides to flow through the nanopore lumen alongthe applied potential. The nanopore may also allow a nucleic acid, suchas DNA or RNA, to be pushed or pulled through the lumen of the nanopore.However, in particular embodiments the nanopore need not allow passageof a double stranded or single stranded nucleic acid. A nanopore used ina particular embodiment can have a minimum lumen diameter of no morethan 10 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm or less. One type ofnanopore is a “protein nanopore” which is a polypeptide or a collectionof polypeptides that forms the small hole to allow passage of nucleotidetriphosphates across a barrier such as a lipid bilayer. Examples ofprotein nanopores include alpha hemolysin nanopore, mycobacteriumsmegmatis porin A (MspA) and variants thereof. Another type of nanoporeis a “solid state nanopore” which is a small hole fabricated through asolid material. The solid material can be, for example, graphene orsilicon.

As used herein the term “nucleotide” is intended to includeribonucleotides, deoxynucleotides or analogs thereof. For example theterm is used herein to generally refer to a nucleoside moiety (whetherribose, deoxyribose, or analog thereof) including a base moiety andoptionally attached to one or more phosphate moieties. Exemplarynucleotides include, but are not limited to, ribonucleotidemonophosphate (sometimes referred to as a ribonucleoside monophosphate),ribonucleotide diphosphate (sometimes referred to as a ribonucleosidediphosphate), ribonucleotide triphosphate (sometimes referred to as aribonucleoside triphosphate), deoxynucleotide monophosphate (sometimesreferred to as a deoxynucleoside monophosphate), deoxynucleotidediphosphate (sometimes referred to as a deoxynucleoside diphosphate) anddeoxynucleotide triphosphate (sometimes referred to as a deoxynucleosidetriphosphate). For clarity when wishing to distinguish RNA componentsfrom DNA components, the term “ribonucleotide” can be used to specifyRNA nucleotides, such as ribouridine triphosphate, riboguanidinetriphosphate, ribocytidine triphosphate or riboadenosine triphosphate;and the term “deoxynucleotide” can be used to specify DNA nucleotides,such as deoxythymidine triphosphate, deoxyguanidine triphosphate,deoxycytidine triphosphate and deoxyadenosine triphosphate. Inparticular embodiments, the nucleotides are ‘extendable’, for example,lacking an extension blocking moiety at the 3′ hydroxyl or at any otherposition on the nucleotide.

As used herein, the term “pyrophosphorolysis” is intended to meanreaction between pyrophosphate and the 3′-nucleotide unit of a nucleicacid to release the nucleotide in the form of the correspondingnucleotide triphosphate. A further product of the reaction is thenucleic acid lacking the corresponding nucleotide (i.e. the reactionconverts a nucleic acid of length n to a nucleic acid of length n−1).The reaction is typically catalyzed by a polymerase and is the reverseof the polymerization reaction carried out by the polymerase understandard biological conditions.

As used herein, the term “pyrophosphorolytic concentration,” when usedin reference to pyrophosphate, is intended to mean a concentration ofpyrophosphate that causes a pyrophosphorolysis reaction to occur at asubstantial level. For example, a pyrophosphorylitic concentration ofpyrophosphate can result in a polymerase displaying a higher rate ofpyrophosphorolysis than polymerization. Thus, a pyrophosphoryliticconcentration of pyrophosphate can result in a substantial reversal ofpolymerization activity that would otherwise be catalyzed by apolymerase.

As used herein, the term “reservoir” is intended to mean a receptacle orchamber for holding or restricting the flow of fluid. A reservoir can befully enclosed, at least temporarily. Alternatively, a reservoir can bepartially enclosed, for example, having one or more opening (e.g. one ormore inlets or outlets). A fluid may flow through a reservoir, forexample, in embodiments where the reservoir is in a flow cell.

As used herein, the term “species” is used to identify molecules thatshare the same chemical structure. For example, a mixture of nucleotidescan include several dCTP molecules. The dCTP molecules will beunderstood to be the same species as each other. Similarly, individualDNA molecules that have the same sequence of nucleotides are the samespecies.

The embodiments set forth below and recited in the claims can beunderstood in view of the above definitions.

The present disclosure provides a method for determining the sequence ofa target nucleic acid. The method can include the steps of (a) providinga target nucleic acid; (b) contacting the target nucleic acid with apolymerase to sequentially remove nucleotide triphosphates from thetarget nucleic acid, wherein the nucleotide triphosphates that areremoved have a variety of different base moieties; and (c)distinguishing the different base moieties for the nucleotidetriphosphates that are removed, thereby determining the sequence of thetarget nucleic acid.

In some embodiments a method for determining the sequence of a targetnucleic acid can be carried out using the steps of (a) providing atarget nucleic acid having two strands; (b) contacting the targetnucleic acid with a polymerase under conditions to sequentially removenucleotides from the first of the two strands by pyrophosphorolysis,thereby sequentially producing nucleotide triphosphates having a varietyof different base moieties; and (c) distinguishing the different basemoieties for the sequentially produced nucleotide triphosphates, therebydetermining the sequence of the target nucleic acid.

An exemplary embodiment is shown in FIG. 1. As shown, a polymerase bindsto a double stranded DNA molecule and, in the presence of excesspyrophosphate, catalyzes a pyrophosphorolysis reaction to releasenucleotide triphosphates from the 3′ end of one of the strands. In thisexample, a deoxyguanidine triphosphate has been produced followed by adeoxythymidine triphosphate. The polymerase is coupled to a nanopore andthe deoxyguanidine triphosphate is in the lumen of the nanopore, whereasthe deoxythymidine triphosphate is in the process of being released intothe nanopore lumen. As such the deoxynucleotide triphosphates enter thenanopore sequentially, in the same order that they were released fromthe DNA strand by the pyrophosphorolytic action of the polymerase. Thedeoxynucleotide triphosphates have a net negative charge due to thetriphosphates and are driven through the nanopore by a potential acrossthe membrane (as indicated by the positive sign on the side of themembrane where pyrophosphorolysis occurs and a negative sign on theopposite side of the membrane). Typically, reagent nucleotidetriphosphates are not present in a pyrophosphorolysis reaction. In somecases, trace amounts of nucleotide triphosphates can be present, but atamounts that do not substantially catalyze a forward primer extensionreaction by polymerase. Thus, in most embodiments the only nucleotidetriphosphates that are substantially present in a pyrophosphorolysisreaction are those produced by the catalytic action of the polymerase onthe nucleic acid.

A similar reaction is exemplified in FIG. 2 except that the templatestrand (i.e. the strand that is not being cleaved by pyrophosphorolysis)is bound to the membrane. In this case, the template strand has acovalently attached sterol moiety (e.g. cholesterol) that binds to thehydrophobic interior of the membrane's lipid bilayer.

A method of the present disclosure can be used with any of a variety oftarget nucleic acids. The target nucleic acid can have a naturallyoccurring structure as found for example in DNA or RNA. DNA naturallycontains monomers having thymine, guanine, cytosine, or adenine bases. ADNA strand that is subjected to pyrophosphorolysis can producedeoxythymidine triphosphate, deoxyguanidine triphosphate, deoxycytidinetriphosphate and deoxyadenosine triphosphate, respectively. DNA can alsocontain some variants of the four nucleotide bases such as 5-methylcytosine, 5-hydroxymethylcytosine and other methylated bases.Deoxynucleotide triphosphates having these variant bases can be producedand/or detected using a method or apparatus set forth herein. Forexample, the presence or absence of methylation on cytosine can bedistinguished to facilitate epigenetic analyses. RNA naturally containsmonomers having uracil, guanine, cytosine, or adenine bases. An RNAstrand that is subjected to pyrophosphorolysis can produce ribouridinetriphosphate, riboguanidine triphosphate, ribocytidine triphosphate orriboadenosine triphosphate, respectively.

A nucleic acid can include non-naturally occurring modifications such asnon-native bases, modifications to the phosphate moieties ormodifications to the sugar moieties. Exemplary non-native bases that canbe included in a nucleic acid, whether having a native backbone oranalog structure, include, without limitation, inosine, xathanine,hypoxathanine, isocytosine, isoguanine, 2-aminoadenine, 6-methyladenine, 6-methyl guanine, 2-propyl guanine, 2-propyl adenine,2-thioLiracil, 2-thiothymine, 2-thiocytosine, 15-halouracil,15-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil,6-azo cytosine, 6-azo thymine, 5-uracil, 4-thiouracil, 8-halo adenine orguanine, 8-amino adenine or guanine, 8-thiol adenine or guanine,8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halosubstituted uracil or cytosine, 7-methylguanine, 7-methyladenine,8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine,3-deazaguanine, 3-deazaadenine or the like.

Non-native bases can be selected, for example, to impart larger orsmaller size, or to impart increased or decreased charge, so as toinfluence the ability of the resulting nucleotide triphosphate analogsto be distinguished by a nanopore or other detection component.Similarly, such bases can be beneficial if selected to impart a desiredrate of pyrophosphorolysis. Non-native bases can be incorporated into anucleic acid using known methods such as amplification or replication ofa template nucleic acid in the presence of the nucleotide analogs. Oneor more of the resulting nucleic acid copies can then be used as targetnucleic acid(s) in apparatus or sequencing method set forth herein.

In particular embodiments, a nucleic acid that is used in a method orapparatus herein will lack one or more of the non-native bases or othernon-native moieties set forth herein. For example, in particularembodiments the methods are not used to remove a terminator nucleotidefrom the 3′ end of a nucleic acid strand. Accordingly, in someembodiments, an apparatus or method may not include any nucleic acid(s)having a terminator nucleotide at its 3′ end. Alternatively oradditionally, in some embodiments, an apparatus or method may notinclude any terminator nucleotide(s).

As exemplified in FIG. 1 and elsewhere herein, a target nucleic acid canbe double stranded DNA, for example, when using a DNA polymerase forpyrophosphorolysis. A heteroduplex, formed between a DNA strand and RNAstrand, can also be used. For example, an RNA polymerase can be used tocatalyze pyrophosphorolysis at the 3′ end of an RNA strand that ishybridized to a DNA template strand, thereby producing ribonucleotidetriphosphates.

A nucleic acid that is used in a method or apparatus herein can beisolated from a biological source and used directly or processed toproduce an amplified or modified product. Alternatively a syntheticnucleic acid can be used, again, directly or after processing.Processing can include, for example, one or more of isolation fromnative components, cleavage to form fragments, amplification (e.g. viaPCR, Rolling Circle or other known techniques), ligation of adaptersequences or tag sequences, tagmentation using a transposon, or “samplepreparation” methods used prior to nucleic acid sequencing techniques.Useful processing techniques are known in the art as set forth, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rdedition, Cold Spring Harbor Laboratory, New York (2001) or in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1998), each of which is incorporated herein byreference.

Examples of sample preparation methods that can be used to processnucleic acids prior to pyrophosphorolysis-based sequencing includemethods that have been developed for whole genome amplification or wholeexome amplification in combination with massively parallel sequencingtechniques. For example, commercially available sample preparationtechniques from Illumina, Inc. (San Diego, Calif.), Life Technologies(Carlsbad, Calif.), 454 Life Sciences (a subsidiary of Roche, Basel,Switzerland) or New England Biolabs (Ipswich, Mass.) can be used.Further useful sample preparation methods that can be used aredescribed, for example, in U.S. Pat. Nos. 6,107,023 or 7,741,463; or USPat. App. Pub. No. 2010/0120098 A1, each of which is incorporated hereinby reference. Targeted sample preparation methods can be used as well inorder to isolate a subset of the sequence content of a complex samplefor subsequent sequencing. Exemplary commercial methods that can be usedfor targeted capture of a subset of nucleic acid fragments include, butare not limited to SureSelect™ kits (Agilent, Santa Clara, Calif.),TruSeq Enrichment Kits (Illumina, Inc., San Diego, Calif.) or Nextera®Enrichment Kits (Illumina, Inc., San Diego, Calif.).

Nucleic acids can be isolated from any of a variety of sourcesincluding, without limitation, a mammal such as a rodent, mouse, rat,rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog,primate, human or non-human primate; a plant such as Arabidopsisthaliana, corn (Zea mays), sorghum, oat (oryza sativa), wheat, rice,canola, or soybean; an algae such as Chlamydomonas reinhardtii; anematode such as Caenorhabditis elegans; an insect such as Drosophilamelanogaster, mosquito, fruit fly, honey bee or spider; a fish such aszebrafish (Danio rerio); a reptile; an amphibian such as a frog orXenopus laevis; a dictyostelium discoideum; a fungi such as pneumocystiscarinii, Takifugu rubripes, yeast, Saccharamoyces cerevisiae orSchizosaccharomyces pombe; or a plasmodium falciparum. Nucleic acids canalso be derived from smaller genomes such as those from a prokaryotesuch as a bacterium, Escherichia coli, staphylococci or mycoplasmapneumoniae; an archae; a virus such as Hepatitis C virus or humanimmunodeficiency virus; or a viroid.

Any of a variety of polymerases can be used in a method or apparatus setforth herein including, for example, protein-based enzymes isolated frombiological systems and functional variants thereof. Reference to aparticular polymerase, such as those exemplified below, will beunderstood to include functional variants thereof unless indicatedotherwise. Examples of useful polymerases include DNA polymerases andRNA polymerases. Exemplary DNA polymerases include those that have beenclassified by structural homology into families identified as A, B, C,D, X, Y, and RT. DNA Polymerases in Family A include, for example, T7DNA polymerase, eukaryotic mitochondrial DNA Polymerase γ, E. coli DNAPol I, Thermus aquaticus Pol I, and Bacillus stearothermophilus Pol I.DNA Polymerases in Family B include, for example, eukaryotic DNApolymerases α, δ, and ε; DNA polymerase ζ; T4 DNA polymerase, Phi29 DNApolymerase, and RB69 bacteriophage DNA polymerase. Family C includes,for example, the E. coli DNA Polymerase III alpha subunit. Family Dincludes, for example, polymerases derived from the Euryarchaeotasubdomain of Archaea. DNA Polymerases in Family X include, for example,eukaryotic polymerases Pol β, pol σ, Pol λ, and Pol μ, and S. cerevisiaePol4. DNA Polymerases in Family Y include, for example, Pol η, Pol iota,Pol kappa, E. coli Pol IV (DINB) and E. coli Pol V (UmuD′2C). The RT(reverse transcriptase) family of DNA polymerases includes, for example,retrovirus reverse transcriptases and eukaryotic telomerases. ExemplaryRNA polymerases include, but are not limited to, viral RNA polymerasessuch as T7 RNA polymerase; Eukaryotic RNA polymerases such as RNApolymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV,and RNA polymerase V; and Archaea RNA polymerase.

The above classifications are provided for illustrative purposes. Itwill be understood that variations in the classification system arepossible. For example, in at least one classification system Family Cpolymerases have been categorized as a subcategory of Family X.Furthermore, polymerases can be classified according to othercharacteristics, whether functional or structural, that may or may notoverlap with the structural characteristics exemplified above. Someexemplary characteristics are set forth in further detail below.

Many polymerases have an intrinsic 3′ to 5′ proofreading exonucleaseactivity which can be useful for some embodiments. Polymerases thatsubstantially lack 3′ to 5′ proofreading exonuclease activity arepreferred in some embodiments, for example, in most sequencingembodiments. Absence of exonuclease activity can be a wild typecharacteristic or a characteristic imparted by a variant or engineeredpolymerase. For example, exo minus Klenow fragment is a mutated versionof Klenow fragment that lacks 3′ to 5′ proofreading exonucleaseactivity. Klenow fragment and its exo minus variant can be useful in amethod or apparatus set forth herein. Polymerases that catalyzepyrophosphorolysis, the direct reversal of polymerization in the sameactive site, are particularly useful.

Depending on the embodiment that is to be used, a polymerase can beeither thermophilic or heat inactivatable. Thermophilic polymerases aretypically useful for high temperature conditions or in thermocyclingconditions such as those employed for polymerase chain reaction (PCR)techniques. Examples of thermophilic polymerases include, but are notlimited to 9° N DNA Polymerase, Taq DNA polymerase, Phusion® DNApolymerase, Pfu DNA polymerase, RB69 DNA polymerase, KOD DNA polymerase,and VentR® DNA polymerase. Most polymerases isolated fromnon-thermophilic organisms are heat inactivatable. Heat inactivation canbe useful to stop a sequencing reaction set forth herein. The reactioncan optionally be re-started by adding a new supply of polymerase to thereaction at the appropriately permissive temperature. Examples of heatinactivatable polymerases are those from phage. It will be understoodthat polymerases from any of a variety of sources can be modified toincrease or decrease their tolerance to high temperature conditions.

An engineered variant of a polymerase having increasedpyrophosphorolysis activity can be used. Exemplary variants are thosethat have been created for use in PCR techniques including, but notlimited to the variants described in U.S. Pat. No. 8,071,536, which isincorporated herein by reference.

A polymerase can be induced to sequentially remove nucleotides from oneof two nucleic acid strands by pyrophosphorolysis in a method set forthherein. The polymerase can be placed under conditions forpyrophosphorolysis to occur. For example, the polymerase can becontacted with a double stranded nucleic acid and a pyrophosphorolyticconcentration of pyrophosphate. Any concentration of pyrophosphate thatcauses a pyrophosphorolysis reaction to occur at a substantial level canbe used including, but not limited to, at least about 100 μMpyrophosphate. Higher concentrations of pyrophosphate can be employed,for example, to drive pyrophosphorolysis at a faster rate. Accordingly,a concentration of at least about 250 μM pyrophosphate, at least about500 μM pyrophosphate, at least about 750 μM pyrophosphate, at leastabout 1 mM pyrophosphate, at least about 2 mM pyrophosphate, at leastabout 5 mM pyrophosphate, at least about 10 mM pyrophosphate, at leastabout 20 mM pyrophosphate or higher can be used.

The ability to alter the rate of pyrophosphorolysis is an advantage fortuning the rate of the sequencing reaction, for example, to accommodatean optimal or desired rate of nucleotide triphosphate detection for agiven detection device. For example, the rate of pyrophosphorolysis canbe decreased by using a lower concentration of pyrophosphate than thoseset forth above. Thus, as an alternative or addition to the thresholdconcentrations set forth above, a maximum concentration of pyrophosphatepresent in a apparatus or method set forth herein can be at most about20 mM pyrophosphate, at most about 10 mM pyrophosphate, at most about 5mM pyrophosphate, at most about 2 mM pyrophosphate, at most about 1 mMpyrophosphate, at most about 750 μM pyrophosphate, at most about 500 μMpyrophosphate, at most about 250 μM pyrophosphate, at most about 100 μMpyrophosphate, or less.

Reagent nucleotide triphosphates are typically absent underpyrophosphorolysis conditions. Thus, in most embodiments the onlynucleotide triphosphates that are substantially present in apyrophosphorolysis reaction are those produced by the catalytic actionof the polymerase on the nucleic acid. If nucleotide triphosphates arepresent under pyrophosphorolysis conditions, the nucleotidetriphosphates will be present at what can be considered a non-catalyticconcentration. A non-catalytic concentration of nucleotide triphosphateis a concentration that is insufficient to allow substantial ordetectable polymerase extension activity to occur. For example, anon-catalytic concentration of nucleotide triphosphate is aconcentration that is substantially below the association bindingconstant for binding of the nucleotide triphosphates to polymerase.Accordingly, pyrophosphorolysis can be carried out by contacting apolymerase with a double stranded nucleic acid in the presence of apyrophosphorolytic concentration of pyrophosphate and no more than anon-catalytic concentration of nucleotide triphosphate.

Exemplary conditions for inducing pyrophosphorolysis that can be usedherein are set forth in Patel et al. Biochem. 30:511-525 (1991), or U.S.Pat. Nos. 7,090,975; 7,914,995 or 8,071,536, each of which isincorporated herein by reference. In several cases these referencesdescribe reactions that also include components used for extension of anucleic acid. It will be understood that extension is not utilized inparticular embodiments of the present disclosure. One or more of thecomponents that are described in Patel et al. Biochem. 30:511-525 (1991)or U.S. Pat. Nos. 7,090,975; 7,914,995 or 8,071,536, including, forexample, components used for polymerase extension reaction, can beexcluded from a method or apparatus set forth herein.

Buffers, salts, metal ions, glycerol, DMSO and other componentstypically included in polymerase storage or reaction mixtures can beincluded in a apparatus or method of the present disclosure, as desired.The quantities and amounts of components to be included will be apparentto those skilled in the art or determinable, for example, via routinetitration techniques.

A beneficial aspect of some embodiments is the ability to stop or pausethe sequencing method by altering the reaction conditions to inhibitpyrophosphorolysis. The sequencing method can then optionally berestarted by altering the reaction conditions to allowpyrophosphorolysis to resume. Any of a variety of the reactionconditions set forth herein, or in the references cited herein, can bealtered between paused pyrophosphorolysis and resumedpyrophosphorolysis, thereby allowing an effective toggle between pausedand active sequencing, respectively.

In particular embodiments, a method of the present disclosure caninclude a step of pausing the sequential production of nucleotidetriphosphates by removing pyrophosphate from contact with a polymerasethat is catalyzing pyrophosphorolysis, followed by a step of resumingthe sequential production of the nucleotide triphosphates by contactingthe polymerase with pyrophosphate. Pyrophosphate can be delivered to areaction using techniques appropriate for the fluid system being usedincluding, for example, pipetting fluid aliquots, movement of fluidboluses under positive or negative pressure (e.g. via pumps or gravity),electrophoresis, isotachophoresis, droplet manipulation (e.g.electrowetting) or the like. Similar fluidic techniques can be used toremove pyrophosphate, for example, by displacement and/or replacementwith a wash solution. Of course, such fluidic techniques can be used toadd or remove other components used in the methods and apparatus setforth herein.

Alternatively or additionally to the fluidic methods set forth above,pyrophosphate can be removed from the reaction by sequestration,degradation or inactivation. For example, physical manipulations can beused such as adsorption to a sequestering agent, or degradation by heat,light or electricity. Chemical methods can be used to modify thestructure or activity of pyrophosphate or to degrade the molecule.Enzymatic methods can also be used such as degradation bypyrophosphatase, as shown for PCR reactions in U.S. Pat. Nos. 4,800,159and 5,498,523 and for gel based sequencing reactions in U.S. Pat. No.4,962,020, each of which is incorporated herein by reference.

Alternatively or additionally to the techniques set forth above,pyrophosphorolysis can be stopped or paused by removing other reactioncomponents. For example, polymerase can be removed from a reaction andoptionally replaced or returned to an active state. For example,polymerase can be removed by fluidic, sequestration, degradation orinactivation methods such as those exemplified above for pyrophosphate.In particular embodiments, a heat sensitive (non-thermophilic)polymerase can be used in a pyrophosphorolysis reaction and then heatinactivated. Similarly, a polymerase can be degraded by chemicalmodification or enzymatic degradation (e.g. via a protease). Whetherdegraded by physical, chemical or enzymatic techniques, the spentpolymerase can be washed away and then pyrophosphorolysis can be resumedby addition of new polymerase to the nucleic acid being sequenced.Polymerase activity can also be toggled by addition and removal ofinhibitors, toggling between permissive and non-permissive temperaturesfor heat stable polymerases, or presence and absence of a sequesteringagent or competitive substrate. Pyrophosphorolysis can also be stoppedand started by denaturation and renaturation, respectively, of thenucleic acid that is being sequenced.

Although methods and apparatus have been exemplified herein forembodiments that use pyrophosphate to drive pyrophosphorolysis, it willbe understood that analogs of pyrophosphate can be used instead. Anexemplary analog is pyrovanadate, which can be used, for example, asdescribed in Akabayov et al. J. Biol. Chem. 286:29146-29157 (2011),which is incorporated herein by reference. As further examples, analogsof pyrophosphate having additional moieties can be used. Generallypyrophosphate analogs are selected that do not entirely inhibitpyrophosphorolysis or passage of the resulting nucleotide triphosphates,or analogs thereof, through a nanopore. However, pyrophosphate analogscan alter characteristics of pyrophosphorolysis and/or nanoporedetection. For example, it may be beneficial to use a pyrophosphateanalog to slow down or speed up pyrophosphorolysis to provide a desiredor optimal detection rate. Similarly, analogs of nucleotidetriphosphates that result when a pyrophosphate analog is used in apyrophosphorolysis reaction can also impart desired characteristics fornanopore detection. For example, moieties that alter charge or size,compared to diphosphate alone, can increase or decrease the rate ofpassage of nucleotide triphosphate analogs through a nanopore, orotherwise alter interactions of the nucleotide triphosphate analogs withthe nanopore, to provide improved sequencing results.

However, in some embodiments a method or apparatus of the presentdisclosure will exclude pyrophosphate having any added moieties. Forexample, pyrophosphate that lacks an optically detectable moiety, suchas a fluorescent moiety, can be used.

In particular embodiments, nucleotide triphosphates are detected usingnanopores. For example, nucleotide triphosphates that are sequentiallyremoved from a nucleic acid via pyrophosphorolysis can be passed througha nanopore for detection. By use of an appropriate nanopore, differentbase moieties of the nucleotide triphosphates can be distinguished toallow sequence detection. Generally an apparatus can be used thatincludes a first and a second compartment separated by a physicalbarrier, wherein the barrier has one or more nanopores. The firstcompartment can include components used for a pyrophosphorolysisreaction. The apparatus can be configured to apply an electric fieldacross the barrier so that nucleotide triphosphates are driven from thefirst compartment through the pore to the second compartment. Theapparatus can be configured for measuring the electronic signature of anucleotide triphosphate passing through the nanopore. Accordingly, auseful apparatus can include an electrical circuit capable of applying apotential and measuring an electrical signal across a barrier andnanopore. The methods may be carried out using a patch clamp or avoltage clamp.

A method of the present disclosure can be carried out using any suitablesystem in which a pore penetrates through a barrier. The barrier in manyembodiments is preferably a lipid bilayer. Lipid bilayers can be madeusing methods known in the art, for example, as described in Montal andMueller Proc. Natl. Acad. Sci. USA 69:3561-3566 (1972) or WO2008/102120, each of which is incorporated herein by reference. Lipidbilayers can be formed from any of a variety of lipids including, butnot limited to, phospholipids, glycolipids, cholesterol and mixturesthereof.

Exemplary nanopores that can be used include, for example, protein basednanopores such as alpha hemolysin nanopore, mycobacterium smegmatisporin A (MspA) and variants thereof. Alpha hemolysin nanopore andvariants of the native nanopore that are particularly useful aredescribed, for example, in US Pat. App. Pub. No. 2011/0229877 A1, orU.S. Pat. Nos. 6,916,665; 7,867,716; 7,947,454; or 8,105,846, each ofwhich is incorporated herein by reference. MspA and variants of thenative nanopore that are particularly useful are described, for example,in US Pat. App. Pub. No. 2012/0055792 A1, which is incorporated hereinby reference. Solid state nanopores can also be useful including, forexample, those described in U.S. Pat. Nos. 6,413,792; 7,444,053; or7,582,490, each of which is incorporated herein by reference.

Detection of nucleotide triphosphates can exploit interaction with ananopore that results in changes to the current flowing through thenanopore in a manner that is specific to each species of nucleotidetriphosphate. For example, a first nucleotide triphosphate species mayreduce the current flowing through the nanopore for a particular meantime period or to a particular extent. A second species of nucleotidetriphosphate can be distinguished by virtue of a different mean timeperiod or a different extent of current alteration when passing throughthe nanopore. Thus, different nucleotide triphosphate species can bedistinguished based on distinctive alterations of the current flowingthrough a nanopore.

Nanopore detection can be carried out using any of a variety ofapparatus known in the art including for example, those described in USPat. App. Pub. Nos. 2011/0229877 A1; or 2012/0055792 A1; or U.S. Pat.Nos. 6,413,792; 6,916,665; 7,867,716; 7,444,053; 7,582,490; 7,947,454;or 8,105,846, each of which is incorporated herein by reference.

A polymerase that is used in an apparatus or method set forth herein canbe present in solution such that it is relatively free to diffuse, atleast within a reaction chamber or it can be relatively limited in itsability to diffuse by being attached to a solid phase support, nanopore,barrier or other component of a method or apparatus set forth herein.Limiting diffusion by attachment can provide an advantage of closelycoupling the point of nucleotide triphosphate production (e.g. apolymerase catalyzing pyrophosphorolysis) with the point of nucleotidetriphosphate detection (e.g. a nanopore through which the nucleotidetriphosphates pass). A polymerase can be attached to a nanopore forexample via a recombinant protein fusion to a subunit of a nanopore,chemical crosslinkage or adapter moiety. Useful methods for attachingpolymerases to nanopores and polymerase-nanopore components are setforth, for example, in US Pat. App. Pub. Nos. 2011/0229877 A1; or2012/0055792 A1; or U.S. Pat. No. 7,947,454, each of which isincorporated herein by reference.

A polymerase can be attached to a bead or other solid support thatresides in a chamber where pyrophosphorolysis occurs. Chemical linkersthat are useful for attaching polymerases to beads or solid supportsinclude those that are commercially available, for example, from ThermoFisher (Rockford, Ill.) or Sigma Aldrich (St. Louis, Mo.) or otherwiseknown in the art.

A polymerase can be attached to a barrier used in a nanopore sequencingapparatus. For example, in embodiments that use a lipid bilayer as thebarrier, a lipophilic moiety can be attached to the polymerase tolocalize the polymerase in proximity to the bilayer due to interactionsbetween the bilayer and lipophilic moiety. Exemplary lipophilic moietiesinclude, but are not limited to, sterols or lipids. A further example ofa lipophilic moiety is a membrane protein (or portion thereof) that canbe attached to a polymerase, for example, via recombinant proteinfusion. Linkages such as those set forth above for beads and other solidsupports can be used to attach a polymerase to a barrier used in solidstate nanopore systems.

A nucleic acid that is sequenced in a method set forth herein or presentin a apparatus of the present disclosure can be in solution such that itis relatively free to diffuse or it can be relatively limited in itsability to diffuse by being attached to a solid phase support, nanopore,barrier or other component of a method or apparatus set forth herein.Attachments similar to those set forth above for polymerases can be usedfor nucleic acids. For example, a sterol, lipid or other lipophilicmoiety can be attached to a nucleic acid to localize it to a lipidbilayer. An example is shown in FIG. 2, where the nucleic acid islocalized to the membrane via a sterol moiety attached to the templatestrand. As exemplified by the figure, the nucleic acid can be attachedvia the template strand, for example, at the 5′ end of the templatestrand. Attachment can also be made at a point on the template that isbetween the location where the polymerase is bound to the template andthe 5′ end of the template.

A lipophilic moiety can be attached to a nucleic acid using methodsknown in the art for attaching other moieties such as biotin orfluorophores. For example, a primer having the lipophilic moiety can beused in an amplification, primer extension, or ligation reaction.Alternatively or additionally, nucleotides having the moiety can be usedin an extension or amplification reaction. If desired, a lipophilicmoiety can be introduced prior to sequencing and during a samplepreparation step, such as those set forth previously herein. Forexample, a targeted sequencing technique can be employed wherein asubset of target nucleic acid having desired sequences are to beselected from a more complex sequence background. In this example, alipophilic moiety can be selectively introduced into the subset oftarget nucleic acids using the targeting technique and this can allowthe targets to be selectively captured by a lipid bilayer, while othernon-targeted sequences are washed away due to not having been modifiedto include the lipophilic moiety.

It can be beneficial in some embodiments to limit diffusion of both thepolymerase and the nucleic acid with respect to a nanopore, for example,using one or more of the attachment means set forth above.

As set forth previously herein, a method of the present disclosure caninclude a step of contacting a target nucleic acid with a polymeraseunder conditions to sequentially remove nucleotides, therebysequentially producing nucleotide triphosphates having a variety ofdifferent base moieties. The variety of different base moieties producedwill depend on the content of the target nucleic acid that is contactedwith the polymerase. For example, DNA typically includes the four commonbases guanine, cytosine, adenine and thymine such thatpyrophosphorolysis will produce deoxyguanidine triphosphate,deoxycytidine triphosphate, deoxyadenosine triphosphate anddeoxythymidine triphosphate. In some cases the target nucleic acid maynot include all four of these base types such that no more than 3, 2 oreven 1 type of deoxynucleotide triphosphate will be produced bypyrophosphorolysis. In some cases, variants of one or more of these fourbase types can be present in the target DNA and accordinglypyrophosphorolysis can produce variant deoxynucleotide triphosphateshaving, for example, methyl, hydroxymethyl or other added moieties.Other variant bases known in the art such as those set froth herein canalso be present in the deoxynucleotide triphosphates produced bypyrophosphorolysis.

Another example is RNA, which typically includes the four common basesguanine, cytosine, adenine and uracil such that pyrophosphorolysis willproduce riboguanidine triphosphate, ribocytidine triphosphate,riboadenosine triphosphate and ribothymidine triphosphate. In some casesthe target nucleic acid may not include all four of these base typessuch that no more than 3, 2 or even 1 type of ribonucleotidetriphosphate will be produced by pyrophosphorolysis. Variant bases, suchas those exemplified herein, for example, with respect todeoxynucleotide triphosphates, or otherwise known in the art, can bepresent in ribonucleotide triphosphates. Generally, the nucleotidetriphosphates produced by pyrophosphorolysis (whether deoxynucleotidetriphosphates or ribonucleotide triphosphates) will include one, atleast two, at least three, at least four or more different base types.

A method of the present disclosure can be carried out under conditionsthat sequentially remove a number of nucleotides from a target nucleicacid, thereby sequentially producing that same number of nucleotidetriphosphates. Furthermore, at least that same number of nucleotidetriphosphates can be distinguished, for example, via passage through ananopore, to allow determination of a sequence having a length that isat least equivalent to the number nucleotides removed from the targetnucleic acid. In particular embodiments the number is at least 1, 2, 3,4, 5, 10, 25, 50, 100, 200, 500, 1000, 10,000 or more up to andincluding the length of the target nucleic acid. Alternatively oradditionally, the number may be no more than 1, 2, 3, 4, 5, 10, 25, 50,100, 200, 500, 1000, or 10,000. The number may be, but need not be,between any two of these values. As set forth previously herein, avariety of techniques can be used to pause pyrophosphorolysis. This canprovide for control of the length of sequence determined usingembodiments of the present methods.

The number of nucleotide triphosphates released by pyrophosphorolysisand/or detected in a method set forth herein may be larger than thenumber of different types of nucleotide triphosphates detected. However,the order and number of the different nucleotide triphosphates detectedcan be correlated with the sequence of the nucleic acid.

In some embodiments it may be beneficial to repeatedly sequence aparticular target nucleic acid. The repetition can be achieved, forexample, by repeatedly processing a target nucleic acid molecule in amethod set forth herein. For example, a method can include the steps of(a) contacting a target nucleic acid with a polymerase to sequentiallyremove nucleotide triphosphates from the target nucleic acid, whereinthe nucleotide triphosphates that are removed have a variety ofdifferent base moieties; (b) distinguishing the different base moietiesfor the nucleotide triphosphates that are removed, thereby determiningthe sequence of the target nucleic acid; (c) regenerating at least aportion of the target nucleic acid; and repeating steps (a) and (b)using the regenerated target nucleic acid. The target nucleic acid canbe regenerated for example by adding nucleotide triphosphates underconditions for the polymerase (or a newly added polymerase) to carry outa polymerization reaction to regenerate at least a portion of a strandof the target nucleic acid that was previously removed bypyrophosphorolysis. Typically pyrophosphate will be substantially absentduring the polymerization reaction.

Alternatively or additionally to the repeated processing embodimentabove, a target nucleic acid can be amplified or copied to createmultiple copies that are processed using a method of the presentdisclosure. A diagrammatic example is shown in FIG. 3. Multiple copiesof a double stranded template nucleic acid are localized to a barrier ina chamber having a nanopore-polymerase fusion (step 1), one of thestrands is captured by the polymerase (step 2), pyrophosphorolysis-basedsequencing occurs (step 3), the template strand, being single stranded,can then be pulled through the nanopore via electric force (step 4)until it is cleared from the chamber where the other copies of thenucleic acid remain (n.b. the other copies remain due to being doublestranded and thus resistant to passage through the nanopore) (step 5),and then another copy of the double stranded template nucleic acid iscaptured to initiate repetition of steps 2 et seq. Any of a variety ofmethods known in the art for amplifying nucleic acids, such as those setforth previously herein, can be used to create the multiple copies ofthe target nucleic acid.

Although the system of FIG. 3 is exemplified for copies of a singletemplate, it will be understood that nucleic acid species havingdifferent sequences can be used similarly. Thus, a variety of differentdouble stranded nucleic acid species can be localized to a barrier in achamber having a nanopore-polymerase fusion (step 1), a strand from afirst species can be captured by the polymerase (step 2),pyrophosphorolysis-based sequencing can occur (step 3), the templatestrand of the first strand can then be pulled through the nanopore viaelectric force (step 4) until it is cleared from the chamber where theother nucleic acid species remain (step 5), and then another doublestranded nucleic acid species can be captured to initiate repetition ofsteps 2 et seq.

The present disclosure also provides an apparatus that includes (a) afluid impermeable barrier separating a first fluid reservoir from asecond fluid reservoir; (b) a nanopore positioned in the fluidimpermeable barrier to form a passage through which a nucleotidetriphosphate can pass from the first fluid reservoir to the second fluidreservoir; and (c) a reaction mix in the first fluid reservoir, thereaction mix including a polymerase, target nucleic acid having twostrands, and pyrophosphorolytic concentration of pyrophosphate. Thecomponents used in the apparatus can be one or more of those exemplifiedabove in the context of various methods. Further components andconfigurations are exemplified below for purposes of illustration.

A fluid impermeable barrier can be configured to separate two reservoirsand to have a nanopore placed in the barrier to provide a fluidconnection between the reservoirs. Exemplary nanopores and barriers areset forth above and in various references set forth above. Generally,the two reservoirs will be in fluid communication via a single nanopore.Thus, nucleotide triphosphates produced in one of the reservoirs willhave one and only one fluid path to the second reservoir. The use of asingle nanopore in this way allows for convenient measurement of eachnucleotide triphosphate that passes from one reservoir to the other dueto changes in electrical properties at the nanopore, barrier and/orreservoirs. However, it is also possible in some embodiments to includemore than one nanopore in the barrier that separates two reservoirs.When multiple nanopores fluidly connect two reservoirs, the passage ofnucleotide triphosphates can be measured at the individual nanoporeusing, for example, optical or electrical measurements that resolve eachnanopore.

A reservoir can create a chamber where fluid remains contained for atleast some of the time. For example, a chamber can be configured to forma well, cavity, compartment etc. that restricts the flow of fluid.Alternatively, a reservoir can be configured for fluid flow. Forexample, the reservoir can be configured as a tube, channel, or flowcell, thereby allowing flow of fluids for convenient delivery andremoval of components used in a sequencing method. In particularembodiments, a first reservoir that contains template nucleic acid,polymerase and pyrophosphate will be configured for fluid flow, whereasthe second reservoir, which is connected to the first chamber via ananopore, can be configured as a chamber. The second reservoir need notbe configured for fluid flow, but optionally can be.

The present disclosure provides multiplex embodiments. For example, thesequences for a plurality of target nucleic acids can be determined inparallel. A multiplex method can include the steps of (a) providing aplurality of target nucleic acids; (b) contacting each of the targetnucleic acids with a polymerase to sequentially remove nucleotidetriphosphates from each target nucleic acid, wherein the nucleotidetriphosphates that are removed have a variety of different basemoieties; and (c) distinguishing the different base moieties for thenucleotide triphosphates that are removed from each nucleic acid,thereby determining the sequences of the target nucleic acids.

A further example of a multiplex method is one that includes the stepsof (a) providing a plurality of target nucleic acids each having twostrands; (b) contacting each of the target nucleic acids with apolymerase under conditions to sequentially remove nucleotides from thefirst of each of the two strands by pyrophosphorolysis, therebysequentially producing nucleotide triphosphates having a variety ofdifferent base moieties; and (c) distinguishing the different basemoieties for the sequentially produced nucleotide triphosphates, therebydetermining the sequence of the target nucleic acids.

A multiplex apparatus can include (a) a plurality of fluid impermeablebarriers that each separate a first fluid reservoir from a second fluidreservoir; (b) a nanopore positioned in each of the fluid impermeablebarriers to form a passage through which a nucleotide triphosphate canpass from the first fluid reservoir to the second fluid reservoir; and(c) a reaction mix in each of the first fluid reservoirs, each of thereaction mixes including a polymerase, target nucleic acid having twostrands, and pyrophosphorolytic concentration of pyrophosphate.

The plexity (i.e. multiplex level) of a method or apparatus can beselected to satisfy a particular use. For example, the number of targetnucleic acids that are processed or present together can be determinedfrom the complexity of the sample to be evaluated. Exemplary complexityestimates for some of the genomes that can be evaluated using methods orapparatus of the present disclosure are about 3.1 Gbp (human), 2.7 Gbp(mouse), 2.8 Gbp (rat), 1.7 Gbp (zebrafish), 165 Mbp (fruit fly), 13.5Mbp (S. cerevisiae), 390 Mbp (fugu), 278 Mbp (mosquito) or 103 Mbp (C.elegans). Those skilled in the art will recognize that genomes havingsizes other than those exemplified above including, for example, smalleror larger genomes, can be used in a method of the invention. Typically anucleic acid sample is fragmented prior to use. The number of fragmentsto be handled in parallel will depend on the complexity of the genome,the average fragment size and the desired coverage. For example, 1×coverage of a human genome (3.1 Gbp) that is fragmented to an averagesize of 1000 nucleotides can be achieved using plexity of 3 millionfragments (i.e. ((3.1 billion/1000)×1)=1 million). Using similarcalculations one can determine that a plexity of 30 million fragments(of 1000 nucleotides each) is sufficient to provide 30× coverage of ahuman genome.

The methods and apparatus set forth herein can be configured at aplexity level sufficient to provide at least 1×, 2×, 5×, 10×, 20×, 30×,50× or more coverage of any of a variety of genomes including, but notlimited to, those exemplified herein. The plexity can be a function ofthe number of various components set forth herein such as the number oftarget nucleic acid fragments as exemplified above. Other componentsthat can be multiplexed include, for example, the number of nanoporesused, the number of polymerases, the number of chambers having a barrierand nanopore etc. The multiplex level of these or other components canbe, for example, at least 2, 5, 10, 100, 1×10³, 1×10⁴, 1×10⁵, 1×10⁶,1×10⁹, or higher. Alternatively or additionally, the multiplex level canbe selected to be no more than 2, 5, 10, 100, 1×10³, 1×10⁴, 1×10⁵,1×10⁶, or 1×10⁹.

Throughout this application various publications, patents and patentapplications have been referenced. The disclosures of these publicationsin their entireties are hereby incorporated by reference in thisapplication in order to more fully describe the state of the art towhich this invention pertains.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the invention. Accordingly, the inventionis limited only by the claims.

What is claimed is:
 1. A method for determining the sequence of a targetnucleic acid, comprising: (a) providing a target nucleic acid having twostrands; (b) contacting the target nucleic acid with a polymerase underconditions to sequentially remove nucleotides from a first strand of thetwo strands by pyrophosphorolysis, thereby sequentially producing singlenucleotide triphosphates having a variety of different base moieties,wherein the conditions to sequentially remove single nucleotidetriphosphates from a first strand of the two strands bypyrophosphorolysis comprise contacting the polymerase with apyrophosphorolytic concentration of pyrophosphate; and (c)distinguishing the variety of different base moieties for thesequentially produced single nucleotide triphosphates, wherein thedistinguishing of the variety of different base moieties for thesequentially produced single nucleotide triphosphates comprises passingthe single nucleotide triphosphates through a nanopore anddistinguishing the single nucleotide triphosphates by detectingvariations in an ionic current flowing through the nanopore, therebydetermining the sequence of the target nucleic acid, wherein thepyrophosphorolysis is paused and then resumed between (b) and (c), toproduce the single nucleotide triphosphates at a desired rate.
 2. Themethod of claim 1, wherein the polymerase is attached to the nanopore.3. The method of claim 1, wherein the nanopore comprises a proteinnanopore that is embedded in a membrane.
 4. The method of claim 3,wherein a second strand of the two strands of the target nucleic acid isattached to the membrane.
 5. The method of claim 1, wherein the nanoporecomprises a solid state nanopore.
 6. The method of claim 1, wherein thepyrophosphorolytic concentration of pyrophosphate comprises aconcentration of at least 100 μM.
 7. The method of claim 1, wherein thepausing the sequential removal of the single nucleotide triphosphates isby removing pyrophosphate from contact with the polymerase and thenresuming the sequential removal of the single nucleotide triphosphatesis by contacting the polymerase with pyrophosphate.
 8. The method ofclaim 1, wherein the variety of different base moieties comprises atleast two different species of base moieties and at most four differentspecies of base moieties.
 9. The method of claim 8, wherein the sequencethat is determined is longer than four nucleotides.
 10. The method ofclaim 1, wherein the variety of different base moieties comprisenaturally occurring adenine, guanine, cytosine or thymine.
 11. Themethod of claim 1, wherein at least one of the variety of different basemoieties comprises a moiety that is non-naturally occurring in DNA orRNA.
 12. The method of claim 1, wherein the target nucleic acid is DNA.13. The method of claim 12, wherein at least one of the variety ofdifferent base moieties comprises a moiety that is non-naturallyoccurring in DNA.
 14. The method of claim 12, wherein at least one ofthe variety of different base moieties comprises 5-methyl cytosine or5-hydroxymethylcytosine.
 15. The method of claim 14, wherein the5-methyl cytosine or 5-hydroxymethylcytosine is distinguished fromcytosine, thereby facilitating an epigenetic analysis.
 16. The method ofclaim 1, wherein the polymerase lacks 3′ to 5′ exonuclease activity. 17.The method of claim 1, wherein the target nucleic acid is a heteroduplexformed by a DNA strand and RNA strand.
 18. The method of claim 1,wherein the conditions to sequentially remove nucleotide triphosphatesfrom one of the two strands by pyrophosphorolysis comprise contactingthe polymerase with a pyrophosphorolytic concentration of apyrophosphate analog.
 19. The method of claim 18, wherein thepyrophosphate analog comprises pyrophosphate having an additionalmoiety.
 20. The method of claim 18, wherein the pyrophosphate analogcomprises pyrovanadate.
 21. The method of claim 1, wherein at least 10nucleotides are sequentially removed from the first strand of twostrands and distinguished by passing through the nanopore.